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INTRODUCTION
Nanotechnology deals with the synthesis, design and manipulation of
nanosized materials. Nanoscale materials are those of size lesser than 100 nm,
possessing one or more dimensions. Some of these materials are natural, while
few are engineered, to attain the desired characteristics. The first reported use of
nanomaterials for human health was over 5,000 years ago in the Indian system of
Ayurveda medicine.
Nanoparticles are three dimensional particles that are present as
aerosol (solid or liquid phase in air), suspension (solid in liquids) or emulsion
(two liquid phases). These particles exhibit completely new or improved
properties based on specific characteristics such as size, distribution and
morphology, when compared with the bulk material they are made of. The most
important and distinct properties of nanoparticles is that they exhibit larger
surface area to volume ratio and unique quantum effects. In the presence of
chemical agents (surfactants), the surface and interfacial properties may be
modified. Indirectly, these agents stabilize the nanoparticles against aggregation,
by conserving particle charge and modifying the outmost layer of the particle.
Depending on the growth history and the lifetime of a nanoparticle, very complex
compositions can be generated. At present, nanotechnology is applied in various
fields like catalysis, electronics, environment, pharmaceutics & biotechnology
(Roco et al., 2010).
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The characteristics of nanoparticles are influenced by several
parameters such as the pH (Udayaprakash et al., 2015), nature of the plant
extract, its concentration, concentration of the metal salt, temperature and contact
time (Dwivedi and Gopal, 2010). On the other hand, the activities of NPs are
mainly dependent on their size and shape.
1.1. ROUTES TO SYNTHESIS OF NANOPARTICLES
There are two approaches in the synthesis of NPs:
1. Top – Down
2. Bottom – Up
The Top – Down approach involves the slicing or successive breaking
of a bulk material while the Bottom – Up approach involves the building up of a
material from the atom, to obtain the nano sized particles. The Top-Down
approach causes internal stress, in addition to surface defects and contaminations.
However, the bottom-up approach promises a better chance to obtain
nanostructures with less defects and more homogeneous chemical composition.
Physical, chemical and biological methods are employed for the synthesis of
nanoparticles. Of these, the physical and chemical methods employ the Top –
Down approach while the biological method employs the Bottom – Up approach.
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Figure 1.1. Routes to synthesis of nanoparticles
1.1.1.Physico chemical methods
The physical methods make use of mechanical forces to convert the
bulk material to smaller granules while the chemical methods involve the use of
reducing agents to the substrate. The obtained nanostructures are further
stabilized by protective agents. Numerous methods are adopted for the
physicochemical methods, of which the most widely employed are Ball milling,
Pyrolysis, Co-precipitation, Sol-gel, Sputtering, Condensation processing, to
name a few. Though the physical and chemical methods enable bulk production,
they involve high costs and cause imperfections. In addition, the NPs synthesized
by these methods lack stability and purity, apart from posing issues related to
toxicity, recycling and disposal.
1.1.2.Biological methods
The obstacles posed by the physical and chemical methods are
overcome to a greater extent, by the biological method. Synthesis of NPs by the
biological methods can be performed either intracellularly or extracellularly by
bacteria, fungi, actinomycetes, algae and plants. In case of microorganisms, the
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intracellular method involves the transport of the metal ions into the cells,
followed by enzyme mediated synthesis of nanoparticles whereas the
extracellular method involves the reduction of the metal ions by enzymes upon
trapping the ions on the cell surface (Zhang et al., 2011). As compared to the size
of extracellularly reduced NPs, the NPs formed inside the organism are smaller.
However, extracellular synthesis of NPs has more applications as compared to
intracellular synthesis since it is devoid of unnecessary adjoining cellular
components from the cell and provides ease of harvesting the NPs. Bacteria have
been most extensively researched for synthesis of nanoparticles because of their
fast growth and relative ease of genetic manipulation. Few bacteria have been
reported to synthesize NPs, i.e., Staphylococcus aureus (Nanda and Saravanan,
2009), Pseudomonas aeruginosa (Husseiny et al., 2007), Brevibacterium casei
(Kalishwaralal et al., 2010), Bacillus sp. (Pugazhenthiran et al., 2009).
Actinomycetes are Gram positive filamentous bacteria that are mainly used for
the production of antibiotics and other metabolites. However, they are not
exploited much in nanotechnology owing to their difficulty in maintenance of the
culture and difficulties in harvesting. Few species of actinomycetes such as
Rhodococcus sp. (Ahmad et al., 2003) Streptomyces sp. and Streptoverticillium
sp. (Prakash et al., 2014) have been reported to synthesize NPs. Among
microbes, fungi are exploited for extracellular synthesis of nanoparticles because
of their enormous secretory components, which are involved in the reduction and
capping of nanoparticles. Alternaria alternata (Sarkar et al., 2012), Fusarium
oxysporum (Ahmad et al., 2003a), Aspergillus fumigatus (Bhainsa and Souza,
2006), Aspergillus flavus (Vigneshwaran et al., 2007), Verticillium sp.
(Mukherjee et al., 2001), Phoma glomerata (Birla et al., 2009), Penicillium
fellutanum (Kathiresan et al., 2009), Hormoconis resinae (Varshney et al., 2009),
Cladosporium cladosporioides (Balaji et al., 2009) are few of the fungal species
reported in synthesis of NPs.
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Figure 1.2. Common methods employed in nanoparticle synthesis
Algae are plant like organisms, capable of performing photosynthesis.
Most of them are eukaryotic, except cyanobacteria. They can be macroscopic or
microscopic. Algae such as Sargassum wightii (Singaravelu et al., 2007),
Sargassum muticum (Azizi et al., 2014), Stoechospermum marginatum (Arockiya
et al., 2012), Tetraselmis kochinensis (Senapati et al., 2012), Cystophora
moniliformis (Prasad et al., 2013) and Turbinaria conoides (Rajeshkumar et al.,
2013) have been explored in nanotechnology, in the recent times.
The mode of synthesis of NPs is dependent on the species chosen for
the study. Of all the sources used for biosynthesis, plants are preferred, due to the
fact that they are easily available and are non-toxic. Moreover, herbal medicines
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are in great demand in the developed as well as developing countries for primary
healthcare because of their wide biological activities, higher safety margins and
lesser costs (Cragg et al., 1997). Plant constituents enable one step process for
synthesis of NPs, are biocompatible, economic, ecofriendly and easy to scale up.
Phytofabrication of nanoparticles, a bottom up approach of synthesis of NPs, is
preferred owing to the stability, efficiency and reduced toxicity of the
phytoconstituents that also serve as reducing and capping agents. The plant
constituents serve as natural capping agents and stabilizers, thereby preventing
the addition of protecting agents as used in other methods. The primary and the
secondary metabolites of the plants are considered to be responsible for the
synthesis of NP, when used in fresh and dry conditions respectively. Few of the
secondary metabolites of plants involved in the synthesis (Dubey et al., 2009;
Huang et al., 2007) are represented in Figure 1.3.
Figure 1.3. Synthesis of nanoparticles from plant metabolites
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Numerous plants such as Avena sativa (Armendariz et al., 2004),
Emblica offcinalis (Ankamwar et al., 2005), Cinnamomum camphora (Huang et
al., 2007), Aloe vera (Chandran et al., 2006), Coriandrum sativum (Narayanan
and Sakthivel, 2008), Carica papaya (Mude et al., 2009), Parthenium
hysterophorus (Parashar et al., 2009), Acanthella elongata (Inbakandan et al.,
2010), Sesuvivm potulacastrum (Nabikhan et al., 2010), Medicago sativa
(Lukman et al., 2011), Trachyspermum ammi and Papaver somniferum
(Vijayaraghavan et al., 2012), Syzygium aromaticum (Vijayaraghavan et al.,
2012a), Nelumbo nucifera (Arokiyaraj et al., 2013), Chrysanthemum indicum
(Arokiyaraj et al., 2014), Stenolobium stans (Prakash et al., 2015), Lonicera
japonica (Kannan et al., 2016), etc., have been exploited for the synthesis of NPs.
Since metal nanoparticles are widely applied in biomedical field, there
is an increasing need for phytofabricated metal nanoparticles which can be scaled
up for commercial applications.
1.2. TYPES OF NANOPARTICLES
The major types of NPs synthesized are organic and inorganic. The
organic NPs include carbon NPs (fullerenes) while the inorganic NPs include
magnetic NPs, noble metal NPs and semiconductor NPs. Inorganic NPs are
mostly preferred as they provide superior material properties with functional
versatility. The applications of metallic NPs vary based on the metal from which
they are synthesized. For example, nanoparticles of iron oxide, gold, silver and
zinc play a major role in medical and biological sector while the Platinum group
metals are employed as fuel cell catalysts. At present, different metallic
nanomaterials are being produced using copper, zinc, titanium, magnesium, gold,
alginate and silver, of which the most extensively studied are those made from
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noble metals, in particular Ag, Pt, Au and Pd (Arangasamy and Munusamy,
2008).
1.2.1. Silver and gold nanoparticles
Syntheses of Ag and AuNPs have been carried out by various
physical, chemical and biological methods, of which the biological methods are
on the rise currently.
The bioreduction of the Ag+ ions by microbes could be associated
with metabolic processes utilizing nitrate by reducing nitrate to nitrile and
ammonium. Enzymes such as nitrate reductase are also considered to play a
major role in the microbe mediated synthesis. Silver nanoparticles are the most
sought-after functionalizing and commercializing nanomaterial due to their
unique properties such as, electric, optical, catalytic, and antimicrobial properties.
The nanoparticles show potent antibacterial activity and a significantly higher
synergistic effect with erythromycin, methicillin, and ciprofloxacin (Devi and
Joshi, 2012). Silver nanoparticles are also reported to possess antifungal, antiinflammatory,
antiviral, anti-angiogenesis and antiplatelet activities, which
confer them the advantage of being developed as alternative products of
commercial importance, for example, as antibiotics against multidrug-resistant
microorganisms. AgNPs as antibacterial agents, are now used extensively in the
fields of medicine (Dar et al., 2013), water treatment (Tolaymat et al., 2010),
molecular imaging (Kohl et al., 2011), diagnosis and treatment of cardiovascular
diseases (Godin et al., 2010), wound healing (Tian et al., 2007), drug delivery
(Meng et al., 2010), clothing (Rafie et al., 2012), purification of water filtering
apparatus (Revina and Egorova, 1998), as cream to prevent infection of burns &
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wounds (Becker, 1999), in sport equipments (Silver, 2003), to treat HIV infection
(Elechiguerra et al., 2005), etc.
In ancient times, colloidal gold was used as a drinkable sol that
exerted curative properties for several diseases (Daniel and Astruc, 2004). Due to
low cytotoxicity of AuNPs (Shukla et al., 2005; Connor et al., 2005) they are
used in diagnostics (Nam et al., 2003), drug delivery (Paciotti et al., 2004; Prow
et al., 2006), cell imaging (Bielinska et al., 2002), immunostaining (Roth, 1996),
biosensing (Penn et al., 2003), and electron microscopy markers (Baschong and
Stierhof, 1998).
1.3. APPLICATIONS OF NANOPARTICLES
Nanoparticles find applications in numerous fields, ranging from
electronics, medicine, energy, treatment of waste, etc., of which the activities
such as antioxidant, antimicrobial, anticancer and catalysis serve the basis for
their applications in medicine and industries.
1.3.1. Antioxidant activity
Reactive oxygen species (ROS) and reactive nitrogen species (RNS)
includes free radicals and other nonradical reactive derivatives. ROS and RNS
includes radicals such as hydroxyl (OH•), superoxide (O2•?), peroxyl (RO2•),
alkoxyl (RO•), hydroperoxyl (HO2•), nitrogen dioxide (NO2•) nitric oxide (NO•)
and lipid peroxyl (LOO•); and non radicals like hydrogen peroxide (H2O2),
nitrogen dioxide (NO2•) hypochlorous acid (HOCl), ozone (O3), singlet oxygen ,
peroxynitrate (ONOO?), nitrous acid (HNO2), lipid peroxide (LOOH), dinitrogen
trioxide (N2O3). Though the radicals are not highly stable, their reactivity is
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stronger than the non radicals (Pham et al., 2008). They tend to become stable by
accepting or donating an electron (Percival, 1998).
Figure 1.4. Applications of nanoparticles
Free radicals are normally generated during normal physiological
processes in living systems. When the free radical generation outnumbers the
antioxidant potency of the host system, oxidative stress occurs, thereby
implicating the pathogenesis of numerous chronic diseases such as Parkinson’s,
Alzheimer, cancer, Atherosclerosis, aging, cardiovascular diseases,
neurodegenerative disorders, etc (Halliwell and Gutteridge, 1999; Sies, 1997).
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Free radical scavenging activity is based on the ability of compounds
to bind with the oxidants by sharing or donating electrons. This can be measured
through several in vitro studies such as hydroxyl, nitric oxide, superoxide,
hydrogen peroxide, DPPH, ABTS radical scavenging assays, etc., which mimic
the role of free radicals as in biological systems.
Figure 1.5.Free radical scavenging activity of antioxidants
Despite the exploration of several natural sources possessing
antioxidant activity, so as to utilize in nutraceuticals ; pharmaceuticals, major
disadvantages such as isolation of specific compound, higher dosage and the
reaction time for scavenging activity are encountered. However, in case of NPs,
these are overcome due to the high surface to volume ratio in NPs that can
suffice lower dosage of the desired compounds. Moreover, NPs possess catalytic
activity, which can enhance the reaction rate, apart from specificity, wherein the
target site can be reached easily.
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1.3.2. Antimicrobial activity
Nosocomial infections due to opportunistic pathogens are a common
cause of mortality among hospitalized patients (Micheal, 1995). Treatment
against infectious microbes is of concern, primarily due to the development of
multidrug resistant strains.
Bacterial infections are the most common in mammals.
Staphylococcus aureus is a Gram positive bacterium that can cause a range of
illnesses, from minor skin infections, such as pimples, impetigo, boils, cellulitis,
folliculitis, carbuncles, scalded skin syndrome, and abscesses, to life-threatening
diseases such as pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock
syndrome, bacteremia, and sepsis. Pseudomonas aeruginosa is a Gram negative,
opportunistic pathogen, which causes infection in the urinary, respiratory ;
gastrointestinal tract, apart from dermatitis, bacteremia and a variety of systemic
infections, specifically in patients with severe burns and the immunosuppressed.
Streptococcus pyogenes, or Group A streptococcus (GAS), is a facultative, Gram
positive coccus which causes numerous infections in humans such as pharyngitis,
tonsillitis, scarlet fever, cellulitis, erysipelas, rheumatic fever, post streptococcal
glomerulonephritis, necrotizing fasciitis, myonecrosis and lymphangitis. The
only known reservoirs for GAS in nature are the skin and mucous membranes of
the human host.
The fungi – Candida albicans, Epidermophyton floccosum and
Microsporum gypseum cause dermatophytosis. C. albicans is the most frequently
isolated etiological agent of candidiasis in humans (Coleman et al., 1998). The
infection is most common in patients with malignant haematological disease and
bone marrow transplant recipients (Warnock, 1998). E. floccosum is the only
pathogen of the two species comprising genus Epidermophyton. The fungi can
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cause Tinea pedis, Tinea cruris, Tinea corporis, and onychomycosis in humans
; animals.
The development of azole-based antifungal drugs has revolutionized
the treatment of many fungal infections, but therapy may still necessitate
application of the highly toxic drug amphotericin B or a combination of drugs.
NPs serve as a better alternative, due to their unique property of high surface to
volume ratio and specificity.
Among the many different metallic nanoparticles, the most effective
action against microorganisms and cancer cells is exhibited by AgNPs (Gong et
al., 2007 and Vaidyanathan et al., 2009). This is because the microbes are
unlikely to develop resistance against silver, as they do against conventional and
narrow-target antibiotics, as the metal attacks a broad range of targets in the
organisms. Hence, the microbes would need to develop mutations simultaneously
for their protection (Sukdeb et al., 2007). The other major metallic NPs proven to
exhibit antimicrobial property are those of Au, Zn ; Fe. The mode of
antimicrobial action is considered to be one or more of the activities depicted in
Figure 1.6.
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Figure 1.6.Antibacterial activity of NPs
1.3.3. Bio-nano catalysis
Nanocatalyst is an emerging innovation that is sought after, for
improving enzyme activity, stability, capability and engineering performances in
bioprocessing applications. Conventionally, they are fabricated by immobilizing
enzymes onto the NPs, such that they act as carriers. Various techniques have
been developed to retain the enzymes on or in the nanomaterials, which include
physical adsorption via electrostatic interactions, hydrophobic interactions,
hydrogen bonding or van der Waals forces, covalent binding, cross-linking of
enzymes or physical entrapment or encapsulation. Other approaches are based on
the functional groups on the surfaces of enzymes such as amino (-NH2),
carboxylate (-COOH), thiol (-SH) and hydroxyl (-OH) groups located in lysine,
arginine, glutamic and aspartic acid residues which can interact with those of
nanocarriers. The functional groups on the nanocarriers have to be introduced by
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surface modifications. The development of novel nanocarriers with unique
functions and characteristics comprises of the following :
(i) Introduction of functional groups on the surface of the nanocarriers for
immobilizing various enzymes or responding to external stimuli,
(ii) Construction of special structures for increasing the surface area, facilitating
substrate diffusion, recycling nanocarriers or confining enzymes inside
nanocages, and
(iii) Improving processability of nanocarriers such as mechanical and thermal
stability (Misson et al., 2015).
However, the use of immobilized enzymes in bioreactors may cause
changes in the native structures of the enzymes, leading to reduction in the
enzyme activity (Talbert and Goddard, 2012). Due to high productivity and
recyclability, the nanoparticles as biocatalysts are a promising candidate for
substrate pre-treatment (Zieminski et al., 2012), biofuel production (Solanki and
Gupta, 2011) and biotransformation (Galanakis, 2012).
1.4. Phyllanthus acidus ; Ruellia tuberosa
Though numerous plants are being exploited for synthesis of
nanoparticles, there is a continuous necessity to synthesize NPs and efficiently
utilize them for medical and industrial applications. However, no reports exist
related with the synthesis of NPs from Phyllanthus acidus and Ruellia tuberosa.
The size and shape of the nanoparticles are influenced by several
parameters at which the synthesis is carried out. Hence, the present study is
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focused on the phytofabrication of Ag and Au NPs, using the leaves and twigs of
a predominant tree – Phyllanthus acidus, belonging to the family Euphorbiaceae
and a herb – Ruellia tuberosa, belonging to Acanthaceae.
Figure 1.7. P. acidus
Rank Scientific name – Common name
Kingdom Plantae – Plants
Subkingdom Tracheobionta – Vascular plants
Superdivision Spermatophyta – Seed plants
Division Magnoliophyta – Flowering plants
Class Magnoliopsida – Dicotyledons
Subclass Rosidae
Order Euphorbiales
Family Euphorbiaceae – Spurge family
Genus Phyllanthus L. – leafflower
Species Phyllanthus acidus (L.) Skeels – Tahitian gooseberry tree
The young leaves of the tree are used as vegetable in Indonesia,
Thailand and India (Prasad, 1986). The tree is of medicinal importance, as it is
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used against several ailments such as psoriasis (Burkill and Mohammed, 2002),
coughs, asthma, bronchitis, soles (Caius, 2003), rheumatism, skin disorders
(Morton et al., 1987), hypertension, respiratory illness (Sousa et al., 2007),
diabetics (Banik et al., 2010) and is hepatoprotective (Lee et al., 2006).
Traditionally, the leaves, bark and root of the tree are used to treat fever (Hadi
and Bremner, 2001). Few reports state the hepatoprotective (Jain and Singhai,
2011), antimicrobial (Jagessar et al., 2008) and nephroprotective (Vidya et al.,
2013) activities of the leaves, in particular.
Figure 1.8. R. tuberosa
Rank Scientific name – Common name
Kingdom Plantae – Plants
Subkingdom Tracheobionta – Vascular plants
Superdivision Spermatophyta – Seed plants
Division Magnoliophyta – Flowering plants
Class Magnoliopsida – Dicotyledons
Subclass Asteridae
Order Scrophulariales
Family Acanthaceae – Acanthus family
Genus Ruellia L. – wild petunia
Species Ruellia tuberosa L. – minnieroot
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Ruellia tuberosa Linn. is a native of Central America and is
introduced into Indian garden as an ornament (Khare, 2007). The herb is used
medicinally in West Indies, Central America, Guiana and Peru (Kritikar and
Basu, 1935). Compounds belonging to flavonoids, steroids, alkaloids and
triterpenoids have been detected in the plant. Antioxidant, gastroprotective,
antimicrobial, anticancer, antinociceptive and anti-inflammatory activities have
been reviewed (Daya et al., 2010). In Siddha system of medicine, the leaves are
given with liquid copal as remedy for gonorrhea and ear diseases (Suseela and
Prema, 2007). The leaves exhibit emetic activity and is employed in the
treatment of bladder stones and Bronchitis (CSIR, 1972). In Suriname’s
traditional medicine system, it is used as anthelmentic and management of joint
pain and strained muscles. In folk medicine, it is used as diuretic and is
considered to possess anti-pyretic, anti-diabetic, antidotal, thirst-quenching agent,
analgesic and anti-hypertensive activities (Chiu and Chang, 1995; Chen et al.,
2006). Additionally, the paste of the leaves are applied on skin diseases, wounds,
boils etc., while the seeds are employed in treating sexual debility,
spermatorrhoea and leucorrhoea (Selvam, 2008). The roots and leaves, used in
form of tea, alleviate retention of urine and are suggested as a remedy to
weakness (Howard, 1983).

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